PROJECT SUMMARY Human bocavirus 1 (HBoV1), an autonomously replicating human parvovirus, causes acute respiratory tract infections in young children. Currently, neither vaccines nor specific therapeutic approaches have been developed for HBoV1-casued acute respiratory tract infections. We have established a cell culture system to produce HBoV1 and used the in vitro culture model of polarized human airway epithelium (HAE) at an air-liquid interface (ALI) to study HBoV1 infection. On the other side, cystic fibrosis (CF) is a genetic disease affecting 30,000 people in USA alone. Utilizing the high apical tropism of airway and large size of the HBoV1 genome, we developed a chimeric parvoviral vector, rAAV2/HBoV1, as an efficient airway transduction vector for CF lung disease gene therapy. rAAV2/HBoV1 is generated through pseudopackaging an oversized recombinant adeno-associated virus 2 (rAAV2) genome (up to 5.9-kb) into the HBoV1 capsid, and it is capable of delivering effective expression of the CF transmembrane conductance regulator (CFTR) to correct the CFTR-specific ion channel deficiency in CF HAE-ALI cultures. More importantly, rAAV2/HBoV1 vector is able to transduce the lungs of ferrets, and CF ferret models closely manifest the phenotype of human CF lung diseases. The co- development of vector and the CF animal model facilitate the preclinical study of CF lung disease gene therapy toward clinical application. Comparative study of HBoV1 infection and rAAV2/HBoV1 transduction in HAE-ALI suggests that rAAV2/HBoV1 vector reassembles the biology of HBoV1 capsid. The transduction of rAAV2/HBoV1 demonstrates unique apical tropism and dependence on cell polarization as does the HBoV1. Although it enters both polarized and non-polarized airway epithelial cells, productive transduction only takes place in polarized HAE. Thus, the development of rAAV2/HBoV1 also provides a powerful means to study the biology of HBoV1 capsid, in which our knowledge is currently limited. In this project, we aim to identify the cellular receptor(s) for HBoV1 capsid entry and to elucidate the mechanisms underlying the HBoV1 receptor- mediated endocytosis and intracellular trafficking in polarized HAE. Our central hypothesis is that HBoV1 utilizes the same receptors on both polarized and non-polarized cells for binding and endocytosis, while the polarization creates an intracellular environment that is in favor of productive transduction. We will use rAAV2/HBoV1 as a tool to identify the glycan-binding attachment and proteinaceous receptors for HBoV1 and to investigate the entry and intracellular trafficking pathways of the HBoV1 capsid in polarized HAE. The success of this project will reveal the key checkpoints that block the interactions between host factors and HBoV1 capsid, which are crucial to the development of antiviral therapeutic strategies and to the optimization of the gene therapy approach to CF lung disease using the rAAV2/HBoV1 vector.